This invention relates to methods to identify test compounds that alter circadian rhythms of mammals, and more specifically that alter the ability of human casein kinase I xcex4 and/or xcex5 to phosphorylate the Human Clock proteins, human Period 1, human Period 2 and human Period 3.
Circadian rhythms generated by endogenous biological pacemakers are present in a number of diverse organisms including humans, fungi, insects and bacteria (Dunlap, J. C. (1999) Cell, 96, 271-290; Hastings, J. W., et al., (1991) in Neural and Integrative Animal Physiology, ed. Prosser, C. L. (New York: Wiley-Liss), pp.435-546; Allada, R., et al., (1998) Cell, 93, 791-804; Kondo, T., et al., (1994) Science, 266, 1233-1236; Crosthwaite, S. K., et al., (1997) Science, 276, 763-769). Circadian clocks are essential in maintaining biological rhythms. They are self-sustaining and constant even under conditions of total darkness but can be entrained by environmental signals such as light and temperature changes. Endogenous clocks control patterns of activity including daily fluctuations in behavior, food intake and sleep/wake cycle as well as physiological changes such as hormone secretion, and fluctuations in body temperature (Hastings, M., (1997) Trends Neurosci. 20, 459-464; Kondo, T., et al., (1993) Proc. Natl. Acad. Sci. USA, 90, 5672-5676.; Reppert, S. M., and Weaver, D. R. (1997) Cell, 89, 487-490).
Genetic and molecular studies in Drosophila have allowed for the elucidation of some of the genes involved in circadian rhythmicity. What has emerged from these studies is a pathway closely auto-regulated and comprised of a transcription/translation-based negative feed back loop (Dunlap, J. C. (1999) Cell, 96, 271-290; Dunlap, J. C. (1996) Annu. Rev. Genet. 30, 579-601; Hall, J. C. (1996) Neuron, 17, 799-802). Two critical components of the central clock are molecules termed Period or PER and Timeless or TIM.
The per locus, first discovered in Drosophila, is a necessary element in controlling circadian rhythms in adult eclosion behavior and locomotor activity (Konopka, R. J., and Benzer, S. (1971) Proc. Natl. Acad. Sci. USA 68, 2112-2116). Missense mutations of PER can either shorten (perS) or lengthen (perL) the period of circadian rhythms, while nonsense mutations (pero) cause arrhythmicity in their behaviors (Hall, J. C. (1995) Trends Neurosci. 18, 230-240). In the suprachiasmatic nuclei (SCN) of mammals, three PER homologues, per1, per2, and per3 have been identified. The protein products of these mammalian genes share several regions of homology to each other (Zylka, M. J., et al., (1998) Neuron 20, 1103-1110; Albrecht, U., et al., (1997) Cell 91, 1055-1064.). Per mRNA and protein levels oscillate during the daily cycle, but only PER1 and PER2 oscillate in response to light (Zylka, M. J., et al., (1998) Neuron 20, 1103-1110., Shearman, L. P., et al., (1997) Neuron 19, 1261-1269).
All PER proteins contain a protein/protein interacting region called the PAS domain that is necessary for dimer formation (Zylka, M. J., et al., (1998) Neuron 20, 1103-1110.). Another PAS containing protein, TIM was isolated by a yeast two-hybrid genetic screen using PER as a bait (Gekakis, N., et al., (1995) Science 270, 811-815). As PER protein levels increase, PER forms heterodimers with TIM. TIM/PER heterodimer formation is required for PER regulation because mutations in tim, cause a loss in circadian rhythm which is accompanied by a loss of per mRNA oscillation and the inability of PER to undergo nuclear translocation (Sangoram, A. M., et al., (1998) Neuron 21, 1101-1113; Zylka, M. J., et al., (1998) Neuron 21, 1115-1122).
Recently, several additional molecular components of circadian rhythmicity including CLOCK and BMAL/MOP3 have been discovered using genetic screening and biochemical characterization (Gekakis, N., et al., (1998) Science 280, 1564-1569; King, D. P., et al., (1997) Cell 89, 641-653; Allada, R., et al., (1998) Cell 93, 791-804).
Subsequent studies shed light on how PER is regulated at transcriptional levels. CLOCK and BMAL/MOP3, both contain basic-helix-loop-helix domain, a PAS domain, and form heterodimers to each other. Once PER is transcribed, translated and accumulated, PER translocates to the nucleus and binds to CLOCK through their common PAS domains and down regulates its own transcription, forming a negative feedback loop (Allada, R., et al., (1998) Cell 93, 791-804; Darlington, T. K., et al., (1998) Science 280, 1599-1603; Hao, H., et al., (1997) Mol. Cell. Biol. 17, 3687-3693; Jin, X., et al., (1999) Cell 96, 57-68.).
In addition, PER is modified and regulated at post-translational levels. Both PER and TIM appear to undergo phosphorylation which is effected by circadian oscillation (Edery, I., et al., (1994) Proc. Natl. Acad. Sci. USA 91, 2260-2264; Lee, C., et al., (1998) Neuron 21, 857-867). A Drosophila kinase termed double time (DBT) was recently cloned (Price, J. L., et al., (1998) Cell 94, 83-95, Kloss, B., et al., (1998) Cell 94, 97-107). Mutations in DBT cause either shortened or lengthened period of the behavioral rhythm. A P-element insertion mutation in DBT abolishes the circadian oscillations of PER in larval brain, indicating that DBT is an essential component of the Drosophila clock. PER accumulates in these mutants to high levels and is hypophosphorylated. DBT has not been shown to directly phosphorylate PER. CKIxcex5 is a closely related homologue of DBT in mammals (Kloss, B., et al., (1998) Cell 94, 97-107). CKIxcex5 and DBT are 86% homologus at the amino acid level in the kinase domain. hCKIxcex5, first identified by Fish et al, is one of several CKI isoforms (xcex1, xcex2, xcex3, xcex4) which has serine/threonine protein kinase activity (Fish, K. J., et al., (1995) J. Biol. Chem. 270, 14875-14883; Rowles, J., et al., (1991) Proc. Natl. Acad. Sci. USA 88, 9548-9552). CKIs are involved in regulation of cellular DNA metabolism. Saccharomyces mutants with defective a HRR25 gene, a homologue to mammalian CKI, show sensitivity to double-stranded DNA breaks (Hoekstra, M. F., et al., (1991) Science 253, 1031-1034). Several in vitro substrates for hCKI have been identified which include RNA polymerases I and II, p53, IkBxcex1, and simian virus 40 large T antigen. However, very little evidence exist which correlates hCKI phosphorylation to changes in substrate function, and to date, no clock genes have been shown to be hCKI xcex4 and xcex5 substrates.
Circadian rhythms are controlled by sequential phosphorylation of, and alterations of protein levels of, certain key proteins in the circadian pathway. Period (PER), a central component of the circadian clock pathway, undergoes daily oscillation in abundance and phosphorylation state. PER genes have been identified in Drosophila PER, designated dPER, mouse PER, designated mPER, and human PER, designated hPER. In Drosophila there is only one PER, which has most homology to the PER1 proteins. Both humans and mice have three PERs, designated PER 1, 2 and 3.
The present invention is directed to discovery that hCKI xcex4 and xcex5 phosphorylate human Period proteins and that phosphorylated human Period proteins are degraded. As a result, the present invention is directed to methods to identify test compounds that alter circadian rhythms of mammals, and more specifically, directed to methods for determining the ability of a test compound to alter hCKI xcex4 and xcex5 phosphorylation of a human Period protein. The present invention is also directed to a method for determining the ability of a test compound to alter degradation of a phosphorylated human Period protein. The present invention is also directed to a method for determing the ability of a test compound to selectively alter phosphorylation, or alternatively degradation, of one or more human Period proteins relative to its ability to alter phosphorylation, or alternatively degradation, of a different human Period protein and subsequently alter the circadian rhythm of a mnammal.